At the present time antibodies are used almost exclusively to identify molecular and cellular structures that do not reveal themselves otherwise. For example, they are used to distinguish one protein from another, one polysaccharide from another, or one cell from another. The interaction between the antibodies and the "identified" structure may be measured by a variety of methods aimed at making visible the presence of the antibodies.
In some tests, an antibody bound to the structure, cell, or solid phase to be identified is directly made visible. More often, a second antibody is made visible and is directed against the first antibody. In addition, protein A of Staphylococcus aureus, Cowan I strain, is made visible and it binds to the first antibody. Another class of reagents used to make the antibody visible is represented by biotin and avidin which recognize each other and can be used in conjunction with antibodies. Fluorescent material, enzymes, radioactive materials, large particles, such as erythrocytes, or beads have been used to make the antibody visible directly or indirectly.
Each of these procedures has shortcomings and limitations which may be illustrated by the use of fluorescent materials to visualize the antibody. If the amount of antibody bound by a cell is too small the fluorescent reagents fail to detect it. This creates analytical difficulties, particularly when using double laser flow cytometry or fluorometric systems. Further, the fluorescence obtained with any antibody system on tissue sections is not very bright. This difficulty may be overcome by using enzymes coupled to the antibody. However, multiple steps in the staining process are always required.
Solutions to the problem have included the use of fluorescent bacteria or fluorescent beads. The former have a limited use in fluorescence analysis due to difficulties in removing unbound excess bacteria and also due to a significant change in the light scattering profile of the cell surface, making impossible the proper use of flow cytometry. The latter tend to stick to cells, are difficult to produce or too expensive, and they are also big enough to cause changes in the light scattering profile of the cell surface. Moreover, in fluorometric systems that require filtration and washing, the excess particles or bacteria are difficult, if not impossible, to remove. The use of reagents such as bacteria or coloidal gold for stained, fixed preparations of cells is limited to microscopy. Accordingly, there has been a need for another class of reagents that will combine the advantages of antibodies with those of particles that can be used for flow cytometry for tissue sections or for solid phase assays for soluble materials. Such solid phases may be those used in kits for measuring antigens or antibodies in solutions, either directly or in competitive assays. It has not been possible to have a reagent that combined all the advantages of antibodies, such as penetrability and specificity, with those of particles, such as visibility. In fact, there was a need for a reagent that works like an antibody but is much larger in size, although not so large as to obscure the structures to be identified. Such a reagent must remain out of some structures, such as fixed cells or tissues, and excess reagent must be readily removed.
It is likely that bacteriophages have been considered either too small to serve as efficient carriers or to be sticky in a nonspecific way, particularly due to their tail fibers. The only use that bacteriophages have had in identification experiments has been to use them as viruses for their bacterial host or to put antigens on their surface, usually through a mild chemical process of coupling, and then to use antibodies against these antigens to block the ability of the bacteriophage to infect bacteria in bacteriophage neutralization systems. Thus, bacteriophage was used only as living virus in Haimovich, et al., U.S. Pat. No. 3,717,705 and in Young, U.S. Pat. No. 4,104,126. It was not employed as a carrier for antibodies as an identification and staining reagent. It is likely that one skilled in the art has been deterred from using the phage for any other assays with antibodies, for the reasons given above.
Luderer, et al., U.S. Pat. No. 4,282,315, proposed the use of radiolabelled animal viruses to detect receptors on materials that are natural receptors of these viruses or mimic such receptors. Essentially the animal viruses were selected for their natural affinity for certain receptor materials and were then removed and multiplied. This was essentially designed either to identify virus receptors or for hemagglutination purposes, i.e., there was a clear similarity between the receptors for virus and those identified. Bacteriophages, which are bacterial viruses, have not been considered in this category for these obvious reasons. First, they are not used for any hemagglutination or hemagglutination inhibition assays; these are based on characteristics of only certain animal viruses. Second, it is well known that bacteriophages have specialized structures, most with the appearance of a tail, that are used for recognition of the receptor on bacteria, leading to infection. It is well known that once this tail binds to its receptor, even when it is not on bacteria but in solution, the nucleic acid is evacuated and the virus becomes non-infectious. Thus, such a method of selection would have been considered impossible according to the techniques of the Luderer, et al., patent. On the other hand, the bacteriophage would not have been considered for selection for its binding with the head protein since mutations in head proteins are considered lethal for the bacteriophage. This may explain why the Luderer, et al., patent did not consider the bacteriophage as a possible alternative, despite the fact that bacteriophages are much cheaper to produce and are easily generated in very large quantity, e.g., orders of magnitude larger than animal viruses.